We describe an enzymatic method for measuring phosphatidylglycerol (PG) at concentrations as low as 0.2 mumol/L in amniotic fluid. Amniotic fluid (1.5 mL) is centrifuged at 10,000g for 20 min to obtain a lamellar body pellet, which is reconstituted with 0.5 mL of buffer. The PG is measured by a two-step enzymatic scheme. Recovery studies demonstrated that the pellet contains > 97% of the PG present in amniotic fluid. Between-run CVs were 28%, 5.7%, and 2.6% for amniotic fluid controls with means of 0.32, 3.9, and 10.7 mumol/L, respectively (n = 20). The enzymatic procedure was not significantly affected by blood, meconium, bilirubin, or other phospholipids. Lecithin/sphingomyelin ratio (n = 101) and fluorescence polarization (n = 127) compared with log(PG) showed correlation coefficients of 0.832 and -0.866, respectively. This test's ability to detect low concentrations of PG in amniotic fluid may make it a better predictor of fetal lung immaturity than previous methods.